Method for diagnosing cancer by detecting gpc3

ABSTRACT

Provided is a method for diagnosing cancer by detecting a novel cancer marker. Cancer can be diagnosed by detecting soluble glypican 3 in a test sample.

FIELD OF THE INVENTION

The present invention relates to a soluble cancer marker in blood, andspecifically relates to a method for diagnosing cancer by detectingsoluble glypican 3 (GPC3) in a clinical sample.

BACKGROUND OF THE INVENTION

The glypican family has been reported to be present as a new family ofheparan sulfate proteoglycans that are present on cell surfaces. Todate, the 5 types of glypicans (glypican 1, glypican 2, glypican 3,glypican 4 and glypican 5) have been reported as members of the glypicanfamily. These members of the family have a core protein of uniform size(approximately 60 kDa), share specific and well-conserved cysteinesequences, and bind to cell membranes via glycosyl phosphatidyl inositol(GPI) anchors.

Glypican 3 (GPC3) has been known to be involved in cell division, or thecontrol of cell division pattern during development, and that a GPC3gene is highly expressed in hepatic cancer cells. Accordingly, the GPC3gene may be used as a hepatoma marker.

We have previously found that an anti-GPC3 antibody has ADCC activityand CDC activity, and is useful for the therapy of hepatic cancer, andhave applied for a patent (Japanese Patent Application No. 2001-189443).

GPC3 is a membrane-bound protein. It has not been reported that asecretory type GPC3 is present in blood and that the GPC3 protein can beused as a cancer marker.

SUMMARY OF THE INVENTION

We have discovered that glypican 3 (GPC3) is cleaved between the 358tharginine and the 359th serine, and thus hypothesized that a soluble GPC3is secreted in blood of hepatic cancer patients. We then established asandwich ELISA system of GPC3, and revealed the presence of GPC3 proteinin the culture supernatant of HepG2 (human hepatic cancer cells) thathighly express GPC3 gene. Furthermore, we also succeeded in measuring asoluble GPC3 not only in the plasma of a mouse grafted with HepG2, butalso in the sera of human hepatic cancer patients. Since the geneexpression of GPC3 in hepatic cancer is observed at an earlier stagethan that of AFP, which is a cancer marker, it has been considered thatthe detection of GPC3 protein is useful for the diagnosis of cancer.Furthermore, the detection of soluble GPC3 is somewhat difficult byusing anti-GPC3 antibodies that can recognize the C-terminal fragment.It has been inferred that the secretory GPC3 protein dominantlycomprises the N-terminal fragment. Therefore, we speculated thatanti-GPC3 antibodies that recognize the N-terminus can be used to detectsoluble GPC3, thereby completing the present invention.

The expression of GPC3 protein has also been detected in cancer celllines other than hepatic cancer cell lines, such as lung cancer, coloncancer, mammary cancer, prostate cancer, pancreatic cancer, andlymphomas. Hence, GPC3 may possibly be applied to the diagnosis ofhepatic cancer as well as many other cancers.

The present invention is as follows.

-   (1) A method for diagnosing cancer, which comprises detecting a    soluble GPC3 protein in a test sample.-   (2) The method for diagnosing cancer of (1), wherein the soluble    GPC3 protein is a N-terminal peptide of GPC3.-   (3) The method for diagnosing cancer of (2), wherein the N-terminal    peptide of GPC3 is a peptide fragment contained in an amino acid    sequence of GPC3 consisting of the 1st amino acid to the 374th amino    acid, or an amino acid sequence of GPC3 consisting of the 1st amino    acid to the 358th amino acid.-   (4) The diagnosis method of any one of (1) to (3), wherein the test    sample is selected from the group consisting of blood, serum and    plasma.-   (5) The diagnosis method of any one of (1) to (4), wherein the    cancer is hepatic cancer.-   (6) The method of any one of (1) to (5), comprising using an    anti-GPC3 antibody.-   (7) The method of (6), comprising using an anti-GPC3 antibody    immobilized on a carrier and an anti-GPC3 antibody labeled with a    labeling substances.-   (8) The method of (7), wherein the labeling substances are biotin.-   (9) A diagnostic reagent for cancer, comprising an anti-GPC3    antibody.-   (10) The diagnostic reagent of (9), comprising an anti-GPC3 antibody    immobilized on a carrier and an antibody labeled with a labeling    substance.-   (11) The diagnostic reagent of (9) or (10), wherein the cancer is    hepatic cancer.-   (12) The diagnostic reagent of any one of (9) to (11), wherein the    anti-GPC3 antibody recognizes the N-terminal peptide of GPC3.-   (13) A diagnostic kit, comprising an anti-GPC3 antibody, and-   (14) The diagnostic kit of (13), comprising the anti-GPC3 antibody    immobilized on carriers, and an antibody labeled with a labeling    substance.

The present invention will be described in detail as follows.

The present invention relates to a method for detecting cancer bydetecting soluble glypican in a test sample.

The meaning of “detection” includes quantitative detection andqualitative detection. Examples of qualitative detection includemeasurement to simply determine whether or not GPC3 proteins arepresent, measurement to determine whether or not the content of GPC3proteins are greater than the basal level, and measurement to comparethe level of GPC3 proteins in a test sample with that of another sample(e.g., a control sample). Examples of quantitative detection includemeasurement of the concentration of GPC3 proteins and measurement of thelevel of GPC3 proteins.

The test sample includes any samples that may contain GPC3 proteins. Thetest sample is preferably collected from the body of an organism such asa mammal, and further preferably collected from a human. Specificexamples of the test sample include blood, intercellular fluid, plasma,extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid,serum, lymph, saliva and urine. Preferred test samples are blood, serumand plasma. The test sample of the present invention also includes thosederived from a test sample, for example, culture media of cellscollected from the body of an organism.

Cancers to be diagnosed according to the invention include, but is notlimited to, hepatic cancer, pancreatic cancer, lung cancer, coloncancer, mammary cancer, prostate cancer, leukemia and lymphomas,preferably hepatic cancer.

1. Preparation of Anti-GPC3 Antibodies

The anti-GPC3 antibody used in the present invention may be derived fromany origin, and may be of any type (monoclonal or polyclonal) and in anyform, as long as it specifically binds to the GPC3 protein.Specifically, known antibodies such as mouse antibodies, rat antibodies,human antibodies, chimeric antibodies or humanized antibodies can beused.

The antibody may be a polyclonal antibody, but preferably a monoclonalantibody.

Furthermore, anti-GPC3 antibodies to be immobilized on carriers andanti-GPC3 antibodies to be labeled with labeling substances mayrecognize the same epitope on the GPC3 molecule, but preferablyrecognize different epitopes.

Preferably, epitopes to be recognized by the antibody is present on theN-terminal fragments (from the 1st amino acid Met to the 358th Arg, orthe 1st Met to the 374th Lys) of the GPC3 protein.

The anti-GPC3 antibody used in the present invention can be obtained asa polyclonal or monoclonal antibody using known techniques. Inparticular, as the anti-GPC3 antibody, a monoclonal antibody derivedfrom a mammal is preferably used in the present invention. Examples ofthe monoclonal antibody derived from a mammal include an antibodyproduced by a hybridoma and an antibody produced by a host transformedby genetic engineering techniques with an expression vector containingthe antibody gene.

A monoclonal antibody-producing hybridoma can be prepared essentiallyusing known techniques as follows. A hybridoma can be prepared byimmunizing an animal with GPC3 as an immunogen according to a standardimmunization method to obtain immunocytes, which are then fused to knownparent cells by a standard cell fusion method. The fused cells arescreened for monoclonal antibody-producing cells by a standard screeningmethod.

Specifically, monoclonal antibodies can be prepared as follows.

GPC3 to be used as an immunogen for raising antibodies is first obtainedby expressing the GPC3 (MXR7) gene as disclosed by Lage, H. et al (Gene188 (1997), 151-156). Specifically, the gene sequence encoding GPC3 isinserted in a known expression vector system, an appropriate host cellis transformed, and then the intended human GPC3 protein is purified bya known method from the host cells or the culture supernatant.

In addition, naturally occurred GPC3 can also be purified and used.

Next, the purified GPC3 protein is used as an immunogen. Alternatively,a partial peptide of GPC3 can be used as an immunogen. In this case, thepartial peptide can be obtained by chemical synthesis based on the aminoacid sequence of human GPC3; incorporation of a part of a GPC3 gene intoan expression vector; or degradation of native GPC3 with a proteolyticenzyme. The region of GPC3 to be used as the partial peptide is notlimited to any specific regions. To obtain an antibody that recognizesan epitope present on the N-terminal fragment, a peptide ranging fromthe 1st amino acid Met to the 358th Arg or a peptide ranging from the1st Met to the 374th Lys of GPC3 may be used. A peptide that contains anepitope of this region but is smaller than the above peptides can alsobe used.

A mammal to be immunized with an immunogen is not specifically limited,and is preferably selected in consideration of compatibility with apartner cell to be used for cell fusion. For example, rodents such asmice, rats, hamsters or rabbits, or monkeys are generally used.

Animals are immunized with an immunogen according to a known method. Forexample, immunization is performed by injecting a mammalintraperitoneally or subcutaneously with an immunogen. Specifically, theimmunogen is diluted with or suspended in an appropriate volume of PBS(Phosphate-Buffered Saline), physiological saline or the like; mixedwith an appropriate volume of a standard adjuvant such as a Freund'scomplete adjuvant if necessary; emulsified; and then administered tomammals several times every 4 to 21 days. In addition, an appropriatecarrier can also be used upon immunization with an immunogen.Particularly when a peptide fragment with a small molecular weight isused as an immunogen, the peptide is preferably bound to a carrierprotein such as albumin or Keyhole limpet hemocyanin, and then used forimmunization.

Mammals immunized as described above is tested for an increased titer ofa desired antibody in the serum. Subsequently, immunocytes are collectedfrom the mammals, and then subjected to cell fusion. A particularlypreferred immunocyte is a splenocyte.

A mammalian myeloma cell is used as a partner cell to be fused with theabove immunocyte. Examples of a myeloma cell line that is preferablyused herein include various known cell lines such as P3 (P3x63Ag8.653)(J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics inMicrobiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. andMilstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. D.H. et al., Cell (1976) 8, 405-415), SP2/0 (Shulman, M. et al., Nature(1978) 276, 269-270), FO (de St. Groth, S. F. et al., J. Immunol.Methods (1980) 35, 1-21), S194 (Trowbridge, I. S. J. Exp. Med. (1978)148, 313-323) and R210 (Galfre, G. et al., Nature (1979) 277, 131-133).

Cell fusion of the above immunocytes with myeloma cells can beessentially performed according to a known method, for example, themethod of Köhler and Milstein et al (Köhler. G. and Milstein, C.,Methods Enzymol. (1981) 73, 3-46).

More specifically, the above cell fusion is performed in a standardnutrition culture medium in the presence of, for example, a cell-fusionaccelerator. A cell-fusion accelerator includes, for example,polyethylene glycol (PEG), Sendai virus (HVJ) or the like. If desired,an auxiliary such as dimethylsulfoxide can also be added to furtherenhance the fusion efficiency.

Any ratio of immunocytes to myeloma cells may be set for use herein. Forexample, it is preferable that the number of immunocytes be 1 to 10times greater than that of myeloma cells. As a medium to be used for theabove cell fusion, for example, RPMI1640 medium or MEM medium which isappropriate for the growth of the above myeloma cell line, or otherstandard media that are used for this type of cell culture can be used.Moreover, a serum fluid such as fetal calf serum (FCS) can be used incombination therewith.

Cell fusion is performed by thoroughly mixing certain amounts of theabove immunocytes and myeloma cells in the above medium, adding PEG(e.g., with an average molecular weight of approximately 1000 to 6000)solution (at a concentration of 30 to 60% (w/v)) pre-heated atapproximately 37° C., and then mixing the solution, so as to form fusedcells (hybridomas). Subsequently, reagents for cell fusion or the likethat is unfavorable for the growth of the hybridomas is removed byadding an appropriate medium successively, removing the supernatant bycentrifugation, and repeating these steps.

The thus obtained hybridomas are selected by culturing the hybridomas ina standard selective medium such as HAT medium (a medium containinghypoxanthine, aminopterin and thymidine). Culture in the above HATmedium is continued for a time period sufficient to kill the non-fusedcells (other than the desired hybridoma) (typically several days toseveral weeks). Subsequently, a standard limiting dilution method isconducted, so that hybridomas that produce the intended antibody arescreened and monocloned.

Screening and monocloning a hybridoma producing a desired antibody maybe performed by a screening method based on a known antigen-antibodyreaction. For example, the antigen is bound to a carrier such aspolystyrene beads or the like or a commercially available 96-wellmicrotiter plate, and the culture supernatant of the hybridomas is addedto the plate to react with the antigen. After the carrier is washed, anenzyme-labeled secondary antibody or the like is added to determinewhether or not an antibody reacting with the immunogen is contained inthe culture supernatant. Hybridomas producing the intended antibodiescan be cloned by the limiting dilution method or the like. Antigens usedfor immunization may be used in this screening. To obtain antibodiesagainst the N-terminal fragment of GPC3, the N-terminal fragment may beused as an antigen for screening.

In addition to the above method where a hybridoma is obtained byimmunizing non-human animals with the antigen, desired human antibodieshaving binding activity to GPC3 can also be obtained by sensitizing invitro human lymphocytes with GPC3, and causing the sensitizedlymphocytes to be fused to human-derived myeloma cells having apermanent division potential (see Japanese Patent Publication (Kokoku)No. 1-59878 B (1989)). Alternatively, GPC3 antigen can be administratedto a transgenic animal having all the repertories of a human antibodygene to obtain anti-GPC3 antibody-producing cells, and then humanantibodies against GPC3 may be obtained from immortalized anti-GPC3antibody-producing cells (see International Patent Publication Nos.WO94/25585, WO93/12227, WO 92/03918 and WO 94/02602).

The thus prepared hybridoma producing a monoclonal antibody can bepassage-cultured in a standard medium, or can be stored for a longperiod in liquid nitrogen.

One example of a method employed to obtain monoclonal antibodies fromthe hybridoma involves culturing the hybridoma according to a standardmethod and obtaining monoclonal antibodies in the culture supernatant.Another method involves administrating the hybridoma to a compatiblemammal and obtaining monoclonal antibodies from its ascites. The formermethod is suitable to obtain highly purified antibodies, while thelatter method is suitable for the mass production of antibodies.

In the present invention, a recombinant monoclonal antibody produced bygenetic engineering techniques can also be used as a monoclonalantibody. The recombinant monoclonal antibody is prepared by cloning theantibody gene from the hybridoma, incorporating the gene into anappropriate vector, introducing the vector into a host, and then causingthe host to produce the recombinant monoclonal antibody (e.g., seeVandamme, A. M. et al., Eur. J. Biochem. (1990) 192, 767-775, 1990).Specifically, mRNA encoding the variable (V) region of an anti-GPC3antibody is isolated from a hybridoma producing the anti-GPC3 antibody.Total RNA is isolated by a known method such as a guanidineultracentrifugal method (Chirgwin, J. M. et al., Biochemistry (1979) 18,5294-5299) or AGPC method (Chomczynski, P et al., Anal. Biochem. (1987)162, 156-159). mRNA is then prepared from the total RNA using mRNAPurification Kit (Pharmacia) or the like. In addition, mRNA can also bedirectly prepared using QuickPrep mRNA Purification Kit (Pharmacia).

cDNA of the antibody V region is synthesized using reverse transcriptasefrom the thus obtained mRNA. For example, cDNA is synthesized using AMVReverse Transcriptase First-strand cDNA Synthesis Kit (SEIKAGAKUCORPORATION) or the like. To synthesize and amplify cDNA, for example,5′-Ampli FINDER RACE Kit (Clontech) and the 5′-RACE method using PCR(Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002,Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) can beemployed.

The desired DNA fragment is purified from the thus obtained PCR product,and then ligated to a vector DNA to prepare a recombinant vector. Thenthe vector is introduced into Escherichia coli or the like, and coloniesare selected, thereby preparing a desired recombinant vector. Thenucleotide sequence of the DNA fragment is confirmed by a known method,such as a dideoxynucleotide chain termination method.

Once DNA encoding the V region of the anti-GPC3 antibody is obtained,this DNA is incorporated into an expression vector containing DNAencoding a desired constant region (C region) of an antibody.

To produce the anti-GPC3 antibody used in the present invention, theantibody gene is incorporated into an expression vector so that the geneis expressed under the regulation of a gene expression control region,for example, an enhancer and a promoter. Next, a host cell istransformed with the expression vector, allowing the host cells toexpress the antibody.

An antibody gene can be expressed by incorporating DNA encoding theantibody heavy chain (H-chain) or DNA encoding the antibody light chain(L-chain) separately into expression vectors, and then simultaneouslytransforming a host cell with the vectors; or by incorporating DNAsencoding the H-chain and the L-chain into a single expression vector,and then transforming a host cell with the vector (see WO 94/11523).

In addition to the above host cells, a transgenic animal can also beused to produce a recombinant antibody. For example, the antibody geneis inserted in a gene encoding a protein (e.g., goat β casein) uniquelyproduced in milk to prepare a fused gene. A DNA fragment containing thefused gene comprising the antibody gene is injected into a goat embryo,and then such embryo is introduced into a female goat. Desiredantibodies can be obtained from the milk produced by the transgenic goatthat has accepted the embryo or its progeny. To increase the milk volumecontaining the desired antibody produced by the transgenic goat,hormones can be administered to the transgenic goat as necessary (Ebert,K. M. et al., Bio/Technology (1994) 12, 699-702).

In the present invention, in addition to the above described antibodies,artificially altered recombinant antibodies such as chimeric antibodiesor humanized antibodies can be used. These altered antibodies can beproduced using a known method.

Chimeric antibodies can be obtained by ligating the DNA encoding theabove antibody V-region to DNA encoding a human antibody C-region,incorporating the product into an expression vector, and thenintroducing the vector into a host to cause the host to produce theantibodies. Using this known method, chimeric antibodies useful in thepresent invention can be obtained.

Humanized antibodies are also referred to as reshaped human antibodies,which are prepared by grafting an antibody CDR (complementaritydetermining region) of a mammal other than a human, such as a mouse, tothe CDR of a human antibody. General gene recombination techniques areknown in the art (see European Patent Application Publication No. EP125023 and WO 96/02576).

Specifically, the DNA sequence that has been designed to ligate a mouseantibody CDR to the framework region (FR) of a human antibody issynthesized by the PCR method using as primers several oligonucleotidesthat have been prepared to have a portion overlapping the terminalregions of both mouse antibody CDR and the framework region (FR) of ahuman antibody (see the method as described in WO 98/13388).

The framework region of a human antibody to be ligated via CDR isselected such that the CDR will form a good antigen binding site. Aminoacids in the framework region in the antibody variable region may besubstituted as required, so that the CDR of a reshaped human antibodyforms an appropriate antigen-binding site (Sato, K. et al., Cancer Res.(1993) 53, 851-856).

C regions derived from a human antibody are used for the C regions of achimeric antibody and a humanized antibody. For example, for theH-chain, Cγ1, Cγ2, Cγ3 or Cγ4C, and for the L-chain, Cκ or Cλ can beused, respectively. In addition, to improve the stability of antibodiesor the production process thereof, the human antibody C-region may bemodified.

A chimeric antibody consists of the variable region of an antibodyderived from a mammal other than a human, and a constant region derivedfrom a human antibody, while a humanized antibody consists of the CDR ofan antibody derived from a mammal other than a human, and the frameworkregion and C region derived from a human antibody. Since theantigenicity of the humanized antibody is expected to be reduced in ahuman body, it is useful as an active component of a therapeutic agentof the present invention.

The antibody used in the present invention is not limited to the entireantigen molecule, but includes a fragment of the antibody or themodified product thereof, as long as it binds to GPC3. Both a bivalentantibody and a monovalent antibody are included. Examples of thefragment of an antibody include Fab, F(ab′)2, Fv, Fab/c having one Faband a complete Fc, and a single chain Fv (scFv) wherein the Fv of theH-chain and the L-chain are linked via an appropriate linker.Specifically, an antibody fragment may be prepared by treating theantibody with an enzyme such as papain or pepsin, or by a method whereingenes encoding these antibody fragments are constructed, introduced intoexpression vectors, and then expressed in appropriate host cells (seee.g., Co., M. S. et al., J. Immunol. (1994) 152, 2968-2976, Better, M. &Horwitz, A. H. Methods in Enzymology (1989) 178, 476-496, AcademicPress, Inc., Plueckthun, A. & Skerra, A. Methods in Enzymology (1989)178, 476-496, Academic Press, Inc., Lamoyi, E., Methods in Enzymology(1989) 121,652-663, Rousseaux, J. et al., Methods in Enzymology (1989)121, 663-669, and Bird, R. E. et al., TIBTECH (1991) 9, 132-137).

scFv is obtained by linking the H-chain V-region and the L-chainV-region of antibodies. In the scFv, the H-chain V-region and theL-chain V-region are linked via a linker, preferably a peptide linker(Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85,5879-5883). The H-chain V-region and the L-chain V-region in scFv may bederived from any of the antibodies described in this specification. As apeptide linker to link the V-regions, for example, any single-strandedpeptide comprising 12 to 19 amino acid residues may be used.

DNA encoding scFv can be obtained as follows. Amplification is performedby the PCR method using as templates the entire or DNA portions encodingdesired amino acid sequences (of DNA encoding the H-chain or the H-chainV-region of the above antibody, and DNA encoding the L-chain or theL-chain V-region), and using a primer pair that specifies both ends.Amplification is then further performed using DNA encoding a peptidelinker portion and a primer pair that specify each end for ligation tothe H-chain and L-chain.

Furthermore, once DNA encoding scFv is prepared, expression vectorscontaining the DNA, and hosts transformed with the expression vectors,can be obtained according to the standard method. In addition, by theuse of the host, scFv can be obtained according to the standard method.

These antibody fragments can be produced by obtaining the genes thereofin a manner similar to the above method, and then causing the expressionof the genes in a host. The “antibody” in the present inventionencompasses such antibody fragments.

Anti-glypican antibodies bound to one of various molecules such as alabeling substance can be used as a modified antibody. The “antibody” inthe present invention also encompasses these modified antibodies. Such amodified antibody can be obtained by chemically modifying the antibodyobtained as above. Methods for antibody modification have beenestablished in the art.

Furthermore, the antibody used in the present invention may be abispecific antibody. The bispecific antibody may have antigen-bindingsites that recognize different epitopes on a GPC3 molecule.Alternatively, one antigen-binding site may recognize GPC3 and the otherantigen-binding site may recognize a labeling substance or the like. Abispecific antibody can be prepared by binding H-L pairs of two types ofantibodies, or by fusing hybridomas producing different monoclonalantibodies. Furthermore, it can also be prepared by genetic engineeringtechniques.

Antibodies can be expressed from the antibody genes constructed asdescribed above by a known method. In the case of mammalian cells, theantibodies can be expressed by operably linking a useful conventionalpromoter, the antibody gene to be expressed, and a polyA signal at the3′ downstream thereof. A promoter/enhancer includes, for example, ahuman cytomegalovirus immediate early promoter/enhancer.

Furthermore, examples of another promoter/enhancer that can be used inthe present invention for antibody expression include aviruspromoter/enhancer such as a retrovirus, a polyoma virus, an adenovirusor a simian virus 40 (SV40), or a promoter/enhancer derived from amammalian cell such as human elongation factor 1a (HEF1a).

When a SV40 promoter/enhancer is used, antibodies can be readilyexpressed by the method of Mulligan et al (Nature (1979) 277, 108), andwhen a HEF1a promoter/enhancer is used, antibodies can be readilyexpressed by the method of Mizushima et al (Nucleic Acids Res. (1990)18, 5322).

In the case of Escherichia coli, a useful conventional promoter, asignal sequence for antibody secretion and an antibody gene are operablylinked, so that the gene can be expressed. A promoter includes laczpromoter and araB promoter. When the lacz promoter is used, the antibodygene can be expressed by the method of Ward et al (Nature (1098) 341,544-546; FASEB J. (1992) 6, 2422-2427), or when the araB promoter isused, the antibody gene can be expressed by the method of Better et al(Science (1988) 240, 1041-1043).

As a signal sequence for the antibody secretion, a pelB signal sequence(Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) may be used when theantibody is produced in the periplasm of Escherichia coli. Afterantibodies produced in the periplasm are isolated, the structure of theantibody is appropriately refolded and used.

A replication origin may be derived from SV40, a polyoma virus, anadenovirus, a bovine papilloma virus (BPV) or the like. Furthermore, toamplify the copy number of the gene in a host cell system, an expressionvector can contain an aminoglycoside transferase (APH) gene, a thymidinekinase (TK) gene, an Escherichia coli xanthine guaninephosphoribosyltransferase (Ecogpt) gene, a dihydrofolate reductase(dhfr) gene or the like as a selection marker.

To produce the antibodies used in the present invention, any expressionsystems such as a eukaryotic cell system or a prokaryotic cell systemcan be used. Examples of eukaryotic cells include animal cells such ascells of established mammalian cell lines or insect cell lines, andfilamentous fungous cells and yeast cells. Examples of prokaryotic cellsinclude bacterial cells such as Escherichia coli cells.

Preferably, antibodies used in the present invention are expressed inmammalian cells such as CHO, COS, myeloma, BHK, Vero or HeLa cells.

Next, a transformed host cell is cultured in vitro or in vivo, so as tocause the host cell to produce the intended antibody. Host cells may becultured according to a known method. For example, DMEM, MEM, RPMI1640,IMDM or the like can be used as a medium. A serum fluid such as fetalcalf serum (FCS) can be used in combination.

The antibodies expressed and produced as described above can be isolatedfrom the cells or host animals, and purified to homogeneity. Isolationand purification of the antibodies to be used in the present inventioncan be performed using affinity columns. An example of a protein Acolumn is Hyper D, POROS, Sepharose F. F. (Pharmacia). Any otherstandard isolation and purification methods for proteins may be used.For example, a chromatography column other than the above affinitycolumn, a filter, ultrafiltration, salting out, dialyses and the likemay be appropriately selected and combined for use, so that antibodiescan be isolated and purified (Antibodies A Laboratory Manual. Ed Harlow,David Lane, Cold Spring Harbor Laboratory, 1988).

2. Detection of GPC3

GPC3 to be detected in the present invention is not specificallylimited, and may be full-length GPC3 or a fragment thereof. When a GPC3fragment is detected, the fragment may be either an N-terminal orC-terminal fragment, and is preferably the N-terminal fragment.Alternatively, the GPC3 to be detected may be a GPC3 protein to whichheparan sulfate or the like is attached, or a GPC3 core protein.

A detection method of GPC3 proteins contained in a test sample is notspecifically limited. Preferably, GPC3 proteins are detected by animmunological method using anti-GPC3 antibodies. Examples of animmunological method include radioimmunoassay, enzyme immunoassay,fluoroimmunoassay, luminescence immunoassay, immunoprecipitation,immuno-nephelometry, Western blot, immunostaining and immunodiffusiontechniques. A preferred detection method is enzyme immunoassay, and aparticularly preferred is enzyme-linked immunosorbent assay (ELISA)(e.g., sandwich ELISA). The above immunological methods such as ELISAcan be performed by a method known to a person skilled in the art.

An example of conventional detection methods using anti-GPC3 antibodiesinvolves immobilizing anti-GPC3 antibodies on a carrier, adding a testsample to the carrier, incubating the carrier to bind GPC3 proteins toanti-GPC3 antibodies, washing, and then detecting GPC3 proteins bound tothe support via anti-GPC3 antibodies to detect a GPC3 protein in a testsample.

Examples of carriers to be used in the present invention includeinsoluble carriers such as insoluble polysaccharides (e.g., agarose orcellulose), synthetic resins (e.g., a silicone resin, a polystyreneresin, a polyacrylamide resin, a nylon resin or a polycarbonate resin)and glass. These carriers can be used in the form of beads or plates. Inthe case of beads, a column or the like can be filled with beads. In thecase of a plate, a multi-well plate (e.g., a 96-well multi-well plate),a biosensor chip or the like can be used. Anti-GPC3 antibodies can bebound to a carrier by any of the conventional methods such as chemicalbinding or physical adsorption. Most of the carriers that can be usedherein may be commercially available.

The binding of anti-GPC3 antibodies with GPC3 proteins is generallyperformed in a buffer. A buffer includes, for example, a phosphatebuffer, a Tris buffer, a citric acid buffer, a borate buffer, acarbonate buffer or the like. Incubation is performed under conditionsthat have already been often used, for example, 1 to 24 hours ofincubation at 4° C. to room temperature. Washing after incubation can beperformed with any solution which does not disturb the binding of GPC3proteins with anti-GPC3 antibodies. For example, a buffer containing asurfactant such as Tween20 is used.

In the detection method of GPC3 proteins of the present invention, acontrol sample can also be set in addition to a test sample containingGPC3 proteins to be detected. Examples of a control sample include anegative control sample containing no GPC3 protein and a positivecontrol sample containing GPC3 proteins. In this case, the resultsobtained from the test sample are compared with the result from thenegative control sample containing no GPC3 protein and the result fromthe positive control sample containing GPC3 proteins, so that GPC3proteins in a test sample can be detected. Moreover, a series of controlsamples are prepared to have serially varied concentrations, thedetection results from each control sample are obtained as numericalvalues, and standard curves are then produced. Based on the standardcurves, the GPC3 protein contained in the test sample can bequantitatively determined from the numerical values obtained from thetest sample.

A preferred embodiment of the detection of a GPC3 protein bound to acarrier via an anti-GPC3 antibody is a detection method using ananti-GPC3 antibody labeled with a labeling substance.

For example, a test sample is allowed to come into contact with ananti-GPC3 antibody immobilized on a carrier. After washing, GPC3 proteinis detected using a labeled antibody that specifically recognizes theGPC3 protein.

Anti-GPC3 antibodies can be labeled by a generally known method. Alabeling substance known by a person skilled in the art, such as afluorescent dye, an enzyme, a co-enzyme, a chemiluminescence substanceor a radioactive substance can be used. Specific examples of a labelingsubstance include a radioisotope (e.g., ³²P, ¹⁴C, ¹²⁵I, ³H and ¹³¹I),fluorescein, rhodamine, dansyl chloride, umbelliferone, luciferase,peroxidase, alkaline phosphatase, β-galactosidase, β-glucosidase,horseradish peroxidase, glucoamylase, lysozyme, saccharide oxidase,microperoxidase and biotin. When biotin is used as a labeling substance,it is preferable to further add avidin bound to an enzyme such asalkaline phosphatase, after the addition of biotin-labeled antibodies.For the binding of labeling substances with anti-GPC3 antibodies, any ofthe known methods such as a glutaraldehyde method, a maleimide method, apyridyl disulfide method or a periodic acid method can be used.

Specifically, a solution containing an anti-GPC3 antibody is added to acarrier such as a plate to allow the anti-GPC3 antibody to beimmobilized on the carrier. After the plate is washed, it is blockedwith, for example, BSA, gelatin, albumin or the like to avoidnonspecific binding of proteins. After the plate is washed again, a testsample is added to the plate. After incubation, the plate is washed, andthen a labeled anti-GPC3 antibody is added. After an appropriateincubation, the plate is washed, and then the labeled-anti-GPC3 antibodyremaining on the plate is detected. Detection can be performed by amethod known by a person skilled in the art. For example, in the case oflabeling with a radioactive substance, the labeled antibody can bedetected by liquid scintillation or an RIA method. In the case oflabeling with an enzyme, a substrate is added, and the consequence ofthe enzymatic reaction of the substrate such as color development can bedetected by a spectrophotometer. Specific examples of a substrateinclude 2,2-azinobis (3-ethylbenzthiazoline-6-sulfonate) diammonium salt(ABTS), 1,2-phenylenediamine (ortho-phenylenediamine) and3,3′,5,5′-tetramethylbenzidine (TME). In the case of labeling with afluorescent substance, the labeled antibody can be detected by afluorometer.

A particularly preferred embodiment of the detection method of a GPC3protein of the present invention makes use of a biotin-labeled anti-GPC3antibody and avidin.

Specifically, a solution containing an anti-GPC3 antibody is added to acarrier such as a plate to allow the anti-GPC3 antibody to beimmobilized on the plate. The plate is washed and blocked with BSA orthe like to avoid nonspecific binding of proteins. The plate is washedagain, and then a test sample is added to the plate. After incubation,the plate is washed, and then a biotin-labeled anti-GPC3 antibody isadded. After appropriate incubation, the plate is washed, and thenavidin conjugated to an enzyme such as alkaline phosphatase orperoxidase is added. After incubation, the plate is washed, a substratecorresponding to the enzyme conjugated to avidin is added, and then GPC3protein is detected using as an indicator an enzymatic change of thesubstrate.

Another embodiment of the detection method of a GPC3 protein of thepresent invention involves using a primary antibody that specificallyrecognizes a GPC3 protein, and a secondary antibody that specificallyrecognizes the primary antibody.

For example, a test sample is allowed to come into contact with ananti-GPC3 antibody immobilized on a support. After incubation andwashing, GPC3 proteins bound after washing are detected using a primaryanti-GPC3 antibody and a secondary antibody that specifically recognizesthe primary antibody. In this case, the secondary antibody has beenpreferably labeled with a labeling substance.

Specifically, a solution containing an anti-GPC3 antibody is added to acarrier such as a plate to allow the anti-GPC3 antibody to beimmobilized to the plate. The plate is washed and blocked with BSA orthe like to avoid nonspecific binding of proteins. The plate is washedagain, and then a test sample is added to the plate. After incubationand washing, a primary anti-GPC3 antibody is added. After appropriateincubation, the plate is washed. Subsequently, a secondary antibody thatspecifically recognizes the primary antibody is added. After appropriateincubation, the plate is washed, and then the secondary antibodyremaining on the plate is detected. The secondary antibody can bedetected by the above-described method.

Another embodiment of the detection method of a GPC3 protein of thepresent invention involves using agglutination reaction. In this method,GPC3 can be detected using carriers sensitized with an anti-GPC3antibody. Any carrier may be used for sensitization of the antibody, aslong as it is insoluble, causes no nonspecific reaction and is stable.For example, latex particles, bentonite, collodion, kaolin orimmobilized sheep erythrocytes can be used. Latex particles arepreferably used. Latex particles used in the invention include, forexample, polystyrene latex particles, styrene-butadiene copolymer latexparticles or polyvinyl toluene latex particles. Polystyrene latexparticles are preferably used. The sensitized particles are mixed with asample, and then the mixture was agitated for a given period of time toobserve agglutination. The higher the concentration of GPC3 antibodiescontained in the sample is, the larger the agglutination degree of theparticles is observed. Thus, GPC3 can be detected by macroscopicobservation of the agglutination. In addition, GPC3 can also be detectedby measuring turbidity resulting from agglutination using aspectrophotometer or the like.

Another embodiment of the detection method of a GPC3 protein of thepresent invention involves a biosensor utilizing the surface plasmonresonance phenomenon. With a biosensor utilizing the surface plasmonresonance phenomenon, the protein-protein interaction can be observed inrealtime in the form of surface plasmon resonance signals using only atrace amount of proteins without labeling. For example, through the useof a biosensor of BIAcore (Pharmacia) or the like, the binding of GPC3proteins to anti-GPC3 antibodies can be detected. Specifically, a testsample is allowed to come into contact with a sensor chip, on which ananti-GPC3 antibody has been immobilized, and then the GPC3 protein boundto the anti-GPC3 antibody can be detected as a change in resonancesignals.

The detection method of the present invention can also be automatedusing various automatic testing systems, so that a large number ofsamples can be tested at one time.

Another object of the present invention is to provide a diagnosticreagent or a kit for detecting GPC3 proteins in a test sample for cancerdiagnosis. The diagnostic reagent or the kit of the invention containsat least an anti-GPC3 antibody. When the diagnostic reagent or the kitis based on the ELISA method, the reagent or the kit may contain acarrier for immobilization of the antibody, or the antibody may havebeen previously bound to the carrier. When the diagnostic reagent or thekit is based on the agglutination method using carriers such as latex,the reagent or the kit may contain carriers having an antibody adsorbedthereon. In addition, the kit may also appropriately contain a blockingsolution, a reaction solution, a reaction stop solution, a reagent fortreating a sample, or the like.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of expression analysis of GPC3 mRNA using GeneChip. FIG. 1A shows the expression of GPC3, and FIG. 1B shows theexpression of alpha-fetoprotein (AFP). NL, CH, LC, WD, MD and PD on thehorizontal axis respectively indicate a normal liver, chronic hepatitissite, liver cirrhosis site, well-differentiated carcinoma,moderately-differentiated carcinoma and poorly-differentiated carcinoma.

FIG. 2 shows an image of CBB staining of purified heparansulfate-attached GPC3 and GPC3 core proteins.

FIG. 3 shows the expression of a GPC3 gene in human hepatic cancer.

FIG. 4 shows the result of western blotting of a soluble core proteinusing an anti-GPC3 antibody.

FIG. 5 shows the principles of sandwich ELISA using an anti-GPC3antibody.

FIG. 6 shows the standard curve of GPC3 sandwich ELISA using M6B1 andM18D4.

FIG. 7 is a schematic view showing the structure of GPC3.

FIG. 8 shows combinations of anti-GPC3 antibodies in ELISA.

FIG. 9 shows standard curves of the GPC3 sandwich ELISA system usingvarious combinations of anti-GPC3 antibodies.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in detail as follows. However,the present invention is not limited by these examples.

In the examples described in the specification of the presentapplication, the following materials were used.

As expression vectors of a soluble GPC3 and a soluble GPC3 core protein,pCXND2 and pCXND3 that had been prepared by incorporating a DHFR geneand a neomycin-resistance gene into pCAGGS were used.

DXB11 cells used herein were purchased from ATCC. For culturing, 5% FBS(GIBCO BRL CAT# 10099-141, LOT# A0275242)/Minimum Essential Medium AlphaMedium (α MEM(+))(GIBCO BRL CAT# 12571-071)/1% Penicillin-Streptomycin(GIBCO BRL CAT#15140-122) was used. For the selection of DXB11 cells,500 μg/mL Geneticin (GIBCO BRL CAT# 10131-027)/5% FBS/a MEM withoutribonucleosides and deoxyribonucleosides (GIBCO BRL CAT# 12561-056) (αMEM(−))/PS was used alone or with supplemented with MTX at a finalconcentration of 25 nM.

HepG2 cells used herein were purchased from ATCC, and maintained in 10%FBS/Dulbecco's Modified Eagle Medium, DMEM) (GIBCO BRL CAT#11995-065)/PS.

Hybridomas were maintained in 10% FBS/RPMI1640/1×HAT media supplement(SIGMA CAT# H-0262)/0.5×BM-Condimed H1 Hybridoma cloning supplement(Roche CAT# 1088947).

EXAMPLE 1 Cloning and Expression Analysis of Human GPC (GPC3) cDNA

Cloning of the Full-Length cDNA Encoding Human Glypican 3 (HereinafterReferred to as GPC3)

The full-length cDNA encoding human GPC3 was amplified by the PCRreaction using the 1st strand cDNA prepared by the standard method fromcolon cancer cell line Caco2 as a template, and an Advantage 2 kit(CLONTECH, Cat. No 8430-1). Specifically, 50 μl of a reaction solutioncontaining 2 μl of the cDNA derived from Caco2, 1 μl of a sense primer(SEQ ID NO: 1), 1 μl of an antisense primer (SEQ ID NO: 2), 5 μl ofAdvantage2 10×PCR buffer, 8 μl of dNTP mix (1.25 mM), and 1.0 μl ofAdvantage polymerase mix was subjected to 35 cycles of a reaction cycleconsisting of 94° C. for 1 minute, 63° C. for 30 seconds and 68° C. for3 minutes. The amplified product (inserted in a TA vector pGEM-T easyusing pGEM-T Easy Vector System I (Promega, Cat. No. A1360)) of the PCRwas sequenced using an ABI3100 DNA sequencer to confirm that cDNAencoding full-length human GPC3 was isolated. The sequence representedby SEQ ID NO: 3 indicates the nucleotide sequence of the human GPC3gene, and the sequence represented by SEQ ID NO: 4 indicates the aminoacid sequence of the human GPC3 protein. SEQ ID NO: 1GATATC-ATGGCCGGGACCGTGCGCACCGCGT: SEQ ID NO: 2GCTAGC-TCAGTGCACCAGGAAGAAGAAGCAC:Expression Analysis of Human GPC3 mRNA Using Gene Chip

The expression analysis of mRNA was performed using GeneChip™ UG95ATarget (Affymetrix) for samples from 24 cases of hepatic cancer(well-differentiated carcinoma: WD; moderately-differentiated carcinoma:MD; poorly-differentiated carcinoma: PD), 16 cases of non hepatic cancer(chronic hepatitis portion: CH; liver cirrhosis portion: LC) and 8 casesof normal liver: NL. These samples were obtained from Graduate School ofMedicine and Faculty of Medicine, the University of Tokyo and theSaitama Cancer Center under informed consent). Specifically, total RNAwas prepared using ISOGEN (Nippon Gene Co., Ltd.) from each of the abovetissues, and then 15 μg of each total RNA was used to perform geneexpression analysis according to the Expression Analysis TechnicalManual (Affymetrix).

As shown in FIG. 1, it was observed that the expression of the mRNA ofthe human GPC3 gene (Probe Set ID: 39350_at) in cancer tissues washigher than that of normal liver tissues in many cases regardless of thedifferentiation stages of hepatic cancer. Furthermore, the mRNAexpression amount of the human GPC3 gene was compared with that of analpha-fetoprotein (Probe Set ID: 40114_at) that is currently the mostfrequently employed as a diagnostic marker of hepatic cancer. As aresult, sufficiently enhanced mRNA expression of GPC3 was observed evenin well-differentiated carcinoma cases where almost no mRNA expressionof the alpha-fetoprotein was observed, and it was revealed that theincidence of enhanced mRNA expression of GPC3 was higher than that ofAFP. Based on the above results, it is thought that GPC3 detection isuseful as an early diagnosis method of hepatic cancer.

EXAMPLE 2 Preparation of Anti-GPC3 Antibodies

Preparation of Soluble Human GPC3

A soluble GPC3 protein lacking the hydrophobic region on the C-terminalside was prepared as a material for preparing anti-GPC3 antibodies.

A soluble GPC3 cDNA expression plasmid DNA was constructed using aplasmid DNA containing full-length human GPC3 cDNA provided by theResearch Center for Advanced Science and Technology, The University ofTokyo. PCR was performed using a downstream primer (5′-ATA GAA TTC CACCAT GGC CGG GAC CGT GCG C-3′ (SEQ ID NO: 5)) designed to eliminate thehydrophobic region (amino acid 564 to amino acid 580) on the C-terminalside and an upstream primer (5′-ATA GGA TCC CTT CAG CGG GGA ATG AAC GTTC-3′ (SEQ ID NO: 6)) containing an EcoR I recognition sequence and aKozak sequence. The thus obtained PCR fragment (1711 bp) was cloned intopCXND2-Flag. The thus prepared expression plasmid DNA was introducedinto a CHO cell line DXB11. A CHO cell line highly expressing solubleGPC3 was obtained by selection using 500 μg/mL Geneticin.

Large-scale culture of the CHO cell line highly expressing soluble GPC3was performed using a 1700 cm² roller bottle. The culture supernatantwas collected and purified. The culture supernatant is applied to DEAEsepharose Fast Flow (Amersham CAT# 17-0709-01). After washing, theproduct was eluted with a buffer containing 500 mM NaCl. Next, theproduct was affinity purified using Anti-Flag M2 agarose affinity gel(SIGMA CAT#A-2220), and eluted by 200 μg/mL FLAG peptide. Afterconcentration using Centriprep-10 (Millipore CAT#4304), the FLAG peptidewas removed by gel filtration using Superdex 200 HR 10/30 (Amersham CAT#17-1088-01). Finally, the protein was concentrated using a DEAEsepharose Fast Flow column, and eluted using a PBS (containing 500 mMNaCl) containing no Tween20 for buffer replacement.

Preparation of Soluble Human GPC3 Core Protein

A cDNA wherein the 495th Ser and 509th Ser were substituted with Ala wasprepared by the assembly PCR method using the above wild type human GPC3cDNA as a template. At this time, a primer was designed so that a Histag was added to the C-terminus. The thus obtained cDNA was cloned intoa pCXND3 vector. The prepared expression plasmid DNA was introduced intoa DXB11 cell line. A CHO cell line highly expressing soluble GPC3 coreprotein was obtained by selection using 500 μg/mL Geneticin.

Large-scale culture was performed using a 1700 cm² roller bottle. Theculture supernatant was collected and purified. The culture supernatantwas applied to Q sepharose Fast Flow (Amersham CAT# 17-0510-01). Afterwashing, the product was eluted using a phosphate buffer containing 500mM NaCl. Next, the product was affinity purified using a Chelatingsepharose Fast Flow (Amersham CAT# 17-0575-01), and eluted with agradient of 10 to 150 mM imidazole. Finally, the product wasconcentrated using a Q sepharose Fast Flow, and then eluted using aphosphate buffer containing 500 mM NaCl.

SDS polyacrylamide gel electrophoresis showed smear bands of 50 to 300kDa and a band of approximately 40 kDa. FIG. 2 shows the result ofelectrophoresis. GPC3 is a proteoglycan having a heparan sulfateaddition sequence on the C-terminus of 69 kDa. The smear bands werethought to be GPC3 modified with heparan sulfate. The amino acidsequencing revealed that the band of approximately 40 kDa contained afragment of the N-terminal side of GPC3, suggesting that GPC3 had beensubjected to some cleavage.

To eliminate antibodies against heparan sulfate in the followinghybridoma screening, a soluble GPC3 core protein was prepared. Namely,two amino acid residues Ser 495 and Ser 509 serving as a heparin sulfateaddition signal sequence were substituted with Ala. A CHO cell linehighly expressing the protein was prepared as above, and then theculture supernatant was subjected to affinity purification utilizing theHis-tag. SDS polyacrylamide gel electrophoresis showed three bands of 70kDa, 40 kDa and 30 kDa. Amino acid sequencing revealed that the band of30 kDa was a fragment on the C-terminal side of GPC3, showing that GPC3had been subjected to some enzymatic cleavages between the 358tharginine and the 359th serine. The band of 30 kDa was not observed inthe heparan sulfate-attached GPC3, possibly because heparan sulfateattached to GPC3 caused the band smear. The fact that GPC3 isenzymatically cleaved at a certain amino acid sequence is a new finding,and its biological significance has not yet been elucidated.

Based on this result, we have hypothesized that GPC3 on the membrane iscleaved also in hepatic cancer patients, and GPC3 of a soluble type issecreted in blood. The gene expression of GPC3 was found to be at ahigher level in early hepatic cancer patients compared with that of AFP,which is a hepatic cancer tumor marker (FIG. 1). Hence, to investigatethe ability of GPC3 as a potential new tumor marker with higher clinicalutility than that of AFP, anti-GPC3 antibodies were prepared andsandwich ELISA systems were constructed as described in Example 2 andthe subsequent examples.

Preparation of Anti-GPC3 Antibodies

Since human GPC3 and mouse GPC3 share high homology of 94% at the aminoacid level, it was thought to be difficult to obtain anti-GPC3antibodies when normal mice are immunized with human GPC3. Thus, MRL/1prmice having autoimmune disease were used for immunization. 5MRL/1pr mice(CRL) were immunized with soluble GPC3. The immunoprotein was preparedat 100 μg/mouse for initial immunization, and then emulsified using FCA(Freund's complete adjuvant (H37 Ra), Difco (3113-60), Becton Dickinson(cat#231131)). The emulsified product was administered subcutaneously. 2weeks later, the protein was prepared at 50 μg/mouse, and thenemulsified using FIA (Freund's incomplete adjuvant, Difco (0639-60),Becton Dickinson (cat#263910)). The emulsified product was administeredsubcutaneously. Subsequently, boosting immunization was performed at1-week intervals 5 times in total. For the final immunization, theprotein was diluted in PBS at 50 μg/mouse, and then administered via thecaudal vein. After the saturation of serum antibody titer against GPC3was confirmed by ELISA using an immunoplate coated with GPC3 coreproteins, P3U1 mouse myeloma cells were mixed with mouse spleen cells toallow for cell fusion in the presence of PEG1500 (Roche Diagnostics,cat#783 641). The fused cells were inoculated on a 96-well cultureplate, and selected using HAT media from the next day, and then theculture supernatant was screened by ELISA. Positive clones weremonoclonalized by the limiting dilution method, followed by expansionculture, and then the culture supernatant was collected. Screening byELISA was performed using the binding activity with GPC3 core proteinsas an indicator, thereby obtaining 6 clones of anti-GPC3 antibodieshaving strong binding ability.

Antibodies were purified using Hi Trap ProteinG HP (AmershamCAT#17-0404-01). The hybridoma culture supernatant was directly appliedto a column. After washing with a binding buffer (20 mM sodium phosphate(pH 7.0)), the antibodies were eluted with an elution buffer (0.1 Mglycine-HCl (pH 2.7)). The eluate was collected in a tube containing aneutralization buffer (1 M Tris-HCl (pH 9.0)), so that the product wasimmediately neutralized. The antibody fraction was pooled and dialyzedagainst 0.05% Tween20/PBS overnight to replace the buffer. NaN₃ wasadded to the purified antibodies at 0.02%, and then the mixture wasstored at 4° C.

Analysis of Anti-GPC3 Antibodies

Mouse IgG sandwich ELISA was performed using goat anti-mouse IgG (gamma)(ZYMED CAT# 62-6600) and alkaline phosphatase-goat anti-mouse IgG(gamma) (ZYMED CAT# 62-6622). The antibody concentration was quantifiedusing a commercially available purified mouse IgG1 antibody (ZYMEDCAT#02-6100) as a standard.

Isotyping of anti-GPC3 antibodies was performed using an ImmunoPureMonoclonal Antibody Isotyping Kit II (PIERCE CAT# 37502) in accordancewith the attached manual. The result of isotyping indicated that all theantibodies were of type IgG1.

Epitope classification of anti-GPC3 antibodies was performed by Westernblotting using the GPC3 core proteins. The soluble GPC3 core proteinswere applied to 10% SDS-PAGE mini (TEFCO CAT#01-075) at 100 ng/lane.After electrophoresis (60 V 30 min, 120 V 90 min), the proteins weretransferred (15 V 60 min) to immobilon-P (Millipore CAT#IPVH R85 10)using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (BIO-RAD).The membrane was briefly washed with TBS-T (0.05% Tween20, TBS),followed by shaking f or 1 hour (at room temperature) or overnight (at4° C.) with TBS-T containing 5% skim milk. After approximately 10minutes of shaking with TBS-T, each of the anti-GPC3 antibodies dilutedto 0.1 to 10 μg/mL with TBS-T containing 1% skim milk was added,followed by 1 hour of shaking. After washing with TBS-T (10 minutes×3times), HRP-anti-mouse IgG antibody (Amersham CAT#NA931) diluted to1/1000 with 1% skim milk-containing TBS-T was added. After 1 hour ofshaking, the membrane was washed with TBS-T (10 minutes×3 times),developed using ECL-Plus (Amersham RPN2132), and imaged on Hyperfilm ECL(Amersham CAT# RPN2103K). FIG. 4 shows the result of Western blotanalysis. The antibodies were classified based on the fact that theantibodies reacting with a band of 40 kDa recognized the epitope on theN-terminus, and the antibodies reacting with a band of 30 kDa recognizedthe epitope on the C-terminus. M6B1, M18D4 and M19B11 antibodies, whichrecognize the N-terminal side, and M3C11, M13B3 and M3B8 antibodies,which recognize the C-terminal side, were obtained. As a result ofanalysis using BIACORE, the KD values of each of the antibodies werebetween 0.2 and 17.6 nM.

EXAMPLE 3 Detection of Soluble GPC3

Mouse Xenograft Model

3,000,000 HepG2 human hepatic cancer cells were grafted subcutaneouslyto the abdominal portion of 6-week-old female SCID mice (Fox CHASEC.B-17/Icr-scid Jcl, CLEA Japan, Inc.) and nude mice (BALB/cA Jcl-nu,CLEA Japan, Inc.). 53 days later (when tumor mass had been sufficientlyformed), the whole blood was collected via the posterior vena cava ofHepG2-grafted SCID mice #1, 3 and 4. Plasma was prepared using a Niproneotube (vacuum blood-collecting tube, NIPRO, NT-EA0205) in the presenceof EDTA-2Na and aprotinin, and then stored at −20° C. by the day ofmeasurement. In addition, the whole blood was collected fromHepG2-grafted SCID mice #2 on day 62 after grafting of HepG2, and fromHepG2-grafted nude mice #1 and 2 on day 66 after grafting through theposterior vena cava. As a control, plasma was also prepared in a similarprocedure from normal SCID mice of the same age.

Sandwich ELISA

To detect soluble GPC3 in blood, a sandwich ELISA system of GPC3 wasconstructed. A 96-well plate was coated with M6B1, and GPC3 bound toM6B1 was detected by M18D4 antibody labeled with biotin. For colordevelopment, AMPAK (DAKO) was used to achieve high sensitivitydetection.

A 96-well immunoplate was coated with the anti-GPC3 antibody dilutedusing a coating buffer (0.1 M NaHCO₃ (pH 9.6), 0.02% (w/v) NaN₃) at 10μg/mL, followed by incubation overnight at 4° C. On the next day, theplate was washed 3 times with 300 μL/well washing buffer (0.05% (v/v)Tween20, PBS), and then 200 μL of dilution buffer (50 mM Tris-HCl(pH8.1), 1 mM MgCl₂, 150 mM NaCl, 0.05% (v/v) Tween20, 0.02% (w/v) NaN₃,1% (w/v) BSA) was added for blocking. The plate was stand at roomtemperature for few hours or at 4° C. overnight, the mouse plasma or theculture supernatant appropriately diluted with a dilution buffer wasadded, and incubated at room temperature for 1 hour. After washing 3times with 300 μL/well of RB, biotin-labeled anti-GPC3 antibodiesdiluted with a dilution buffer at 10 μg/mL were added, and incubated atroom temperature for 1 hour. After washing 3 times with 300 μL/well ofRB, AP-streptavidin (ZYMED) diluted to 1/1000 with a dilution buffer wasadded, and incubated at room temperature for 1 hour. After washing 5times with 300 μL/well washing buffer, color development was performedusing AMPAK (DAKO CAT#K6200) according to the attached protocols.Absorbance was then measured using a microplate reader.

A Biotin Labeling Kit (CAT# 1 418 165, Roche) was used for biotinylationof antibodies. The soluble GPC3 concentration in a sample was calculatedusing a GlaphPad PRISM spreadsheet program (GlaphPad software Inc. ver.3.0). FIG. 5 shows the principles of the sandwich ELISA of this example.

A standard curve was prepared using purified soluble GPC3, so that asystem having a detection limit of several ng/mL could be constructed.FIG. 6 shows the standard curve of the GPC3 sandwich ELISA using M6B1and M18D4. Using this system, detection of GPC3 in the culturesupernatant of the above HepG2 and the mouse sera to which HepG2 humanhepatic cancer cells had been grafted was attempted. The soluble GPC3was detected in the culture supernatant of HepG2 and in the sera of themice to which HepG2 human hepatic cancer cells had been grafted, whilethe soluble GPC3 levels in the control medium and in control mouse serawere below the detection limit. When expressed in terms of theconcentration of purified soluble GPC3, the concentration was 1.2 μg/mLin HepG2 culture supernatant, and 23 to 90 ng/mL in the mouse sera(Table 1). TABLE 1 Measurement of soluble GPC3 concentration inHepG2-grafted mouse plasma (ng/mL) Tumor volume M6B01(N)- M19B11(N)-M6B1(N)- M13B3(C)- M13B3(C)- (mm3) M18D4(N) M18D4(N) BioM3C11(C)BioM18D4(N) BioM3B8(C) HepG2 culture 1190 1736 224 234 <1 supernatantHepG2-grafted SCID 2022 65.4 76.9 <10 <10 <10 mouse #1 HepG2-graftedSCID 1705 71.7 94.8 <10 <10 <10 mouse #2 HepG2-grafted SCID 2257 90.3113.9 <10 <10 <10 mouse #3 HepG2-grafted SCID 2081 87.3 107.3 <10 15.0<10 mouse #4 HepG2-grafted nude 1994 58.7 53.6 19.7 35.5 102.2 mouse #1HepG2-grafted nude 190 & 549 22.9 33.6 <10 11.5 40.6 mouse #2 NormalSCID mouse #1 0 <10 <10 <10 <10 <10 Normal SCID mouse #2 0 <10 <10 <10<10 <10 Normal SCID mouse #3 0 <10 <10 <10 <10 <10Structure of Secretory GPC3

It was investigated whether GPC3 was cleaved between the 358th arginineand the 359th serine and secreted as previously hypothesized. Ifsecretory GPC3 is an N-terminal fragment, it may not be possible todetect this type of GPC3 with a sandwich ELISA using a combination ofthe antibody recognizing the N-terminus and the antibody recognizing theC-terminus. Using 3 types each of the antibodies recognizing theN-terminal fragment and the antibodies recognizing the C-terminalfragment, sandwich ELISA systems with various combinations of theantibodies were constructed. FIG. 7 shows the structure of secretorysoluble GPC3, and FIG. 8 shows the combinations of antibodies. FIG. 9shows the standard curves of the sandwich ELISA. Table 1 shows themeasurement results. As shown in Table 1, secretory GPC3 was detected athigh levels in the culture supernatant of HepG2, and in the sera of themice to which HepG2 human hepatic cancer cells had been grafted using acombination of the antibodies, both of which recognize the N-terminalfragment. On the other hand, the detection results obtained with asystem comprising antibodies recognizing the C-terminal fragment werebelow the detection limit in many mice. Accordingly, it was predictedthat N-terminal fragment would be dominant in the secretory GPC3discovered by the invention.

INDUSTRIAL APPLICABILITY

As shown in these examples, it was shown that GPC3 is highly expressedin hepatic cancer cells, and a portion of GPC3 may be present in bloodin the form of a secretory protein. Since gene expression of GPC3 isobserved in cancer tissues at a stage earlier than that of AFP, ahepatic cancer marker, detection of GPC3 is thought to be useful forcancer diagnosis. The expression of GPC3 was also found in cell lines ofcancer other than hepatic cancer, such as lung cancer, colon cancer,cancer, prostate cancer, pancreatic cancer or lymphomas. Thus, GPC3 mayhave possible applications for diagnosis of cancers other than hepaticcancer.

Moreover, a possibility shown herein was that the N-terminal fragmentcleaved between the 358th arginine and the 359th serine is dominantlypresent in the secretory GPC3. Accordingly, it is thought that theantibody recognizing the N-terminal fragment is useful as an antibodyfor diagnosis. Furthermore, if the antibody recognizing the C-terminalfragment is used as an antibody having ADCC activity and CDC activityfor treating hepatic cancer, it may be able to efficiently reach hepaticcancer cells without being trapped by the secretory GPC3 present inblood.

All publications, patents and patent applications cited herein areincorporated herein by reference in their entirety. It is therefore toreadily be understood by a person skilled in the art that numerousmodifications and variations of the present invention are possiblewithin the scope of the invention without departing from the technicalidea and the scope of the invention as described in the appended claims.The present invention is intended to encompass such modifications andvariations.

1. A method for diagnosing cancer, which comprises detecting a solubleGPC3 protein in a test sample.
 2. The method for diagnosing cancer ofclaim 1, wherein the soluble GPC3 protein is a N-terminal peptide ofGPC3.
 3. The method for diagnosing cancer of claim 2, wherein theN-terminal peptide of GPC3 is a peptide fragment contained in an aminoacid sequence of GPC3 consisting of the 1 st amino acid to the 374thamino acid, or an amino acid sequence of GPC3 consisting of the 1stamino acid to the 358th amino acid.
 4. The diagnosis method of claim 1,wherein the test sample is selected from the group consisting of blood,serum and plasma.
 5. The diagnosis method of claim 1, wherein the canceris hepatic cancer.
 6. The method of claim 1, comprising using ananti-GPC3 antibody.
 7. The method of claim 6, comprising using ananti-GPC3 antibody immobilized on a carrier and an anti-GPC3 antibodylabeled with a labeling substances.
 8. The method of claim 7, whereinthe labeling substances are biotin.
 9. A diagnostic reagent for cancer,comprising an anti-GPC3 antibody.
 10. The diagnostic reagent of claim 9,comprising an anti-GPC3 antibody immobilized on a carrier and anantibody labeled with a labeling substance.
 11. The diagnostic reagentof claim 9, wherein the cancer is hepatic cancer.
 12. The diagnosticreagent of claim 9, wherein the anti-GPC3 antibody recognizes theN-terminal peptide of GPC3.
 13. A diagnostic kit, comprising ananti-GPC3 antibody.
 14. The diagnostic kit of claim 13, comprising theanti-GPC3 antibody immobilized on carriers, and an antibody labeled witha labeling substance.
 15. The diagnosis method of claim 3, wherein thetest sample is selected from the group consisting of blood, serum andplasma.
 16. The diagnosis method of claim 3, wherein the cancer ishepatic cancer.
 17. The diagnosis method of claim 4, wherein the canceris hepatic cancer.
 18. The method of claim 3, comprising using ananti-GPC3 antibody.
 19. The diagnostic reagent of claim 10, wherein thecancer is hepatic cancer.
 20. The diagnostic reagent of any one of claim11, wherein the anti-GPC3 antibody recognizes the N-terminal peptide ofGPC3.